Calcium phosphate microcarriers and microsphers

ABSTRACT

The present invention provides calcium phosphate-based (CaP) microcarriers and their use, for example, in cell culturing systems, chromatography, and implantable biomedical materials.

FIELD OF THE INVENTION

[0001] The invention disclosed herein relates to calcium phosphate (CaP)microcarriers and microspheres and their use, for example, in cellculturing systems, chromatography analysis and processing, andimplantable materials useful for biomedical implants.

BACKGROUND

[0002] Revolutionary advances in biotechnology and genetic engineeringhave created enormous potential for marketing cellular by-products,including for example, proteins, including protein pharmaceuticals suchas interferon, monoclonal antibodies, TPA (Tissue PlasminogenActivator), growth factors, insulin, and cells for transplantation. Thedemand for these products has grown tremendously and will continue to doso, creating a need for efficient methods of producing industrialquantities of cell-derived pharmaceuticals and other products. Further,the demand for efficient methods of analyzing and isolating biologicalproducts through chromatographic technology, and the need to improvebio-implantables continues to grow.

[0003] Research and study of cell structure and morphology arefundamental to continued progress in the diagnosis and treatment ofhuman diseases. Numerous cell products are of vital importancetherapeutically and commercially, including, for example, hormones,enzymes, viral products, vaccines, and nucleic acids. The production ofthese products requires large scale cell culture systems for theirproduction.

[0004] Mammalian cells can be grown and maintained in vitro, but aregenerally anchorage-dependent, i.e., they require a solid surface orsubstrate for growth. The solid substrate is covered by or immersed in anutrient medium particular to the cell type to be cultured. The nutrientmedium and solid substrates are contained in a vessel and provided withan adequate supply of oxygen and carbon dioxide to support cell growthand maintenance. Cell cultures may be batch systems, in which nutrientsare not replenished during cultivation although oxygen is added asrequired; fed batch systems, in which nutrient and oxygen are monitoredand replenished as necessary; and perfusion systems, in which nutrientand waste products are monitored and controlled (Lubiniecki, Large ScaleMammalian Cell Culture Technology, Marcel Dekker, Inc., New York, 1990).

[0005] The primary commercial systems used for mammalian cell cultureuse solid matrix perfusion and microcarrier bead systems (Lubineicke,supra). The solid matrix perfusion systems utilize glass columns packedwith glass beads or helices, which form a matrix as the solid substratefor cell growth. Once cells have attached to the matrix, medium iscontinuously recycled from a storage vessel for support of cell growthand maintenance. A similar perfusion system uses hollow fibers as thesolid matrix instead of beads.

[0006] In microcarrier systems, small spheres are fabricated, forexample, from an ion exchange gel, dextran, polystyrene, polyacrylamide,or collagen-based material. These materials have been selected forcompatibility with cells, durability to agitation and specific gravitiesthat will maintain suspension of the microcarriers in growth mediums.Microcarriers are generally kept in suspension in a growth medium bygently stirring them in a vessel. Microcarrier systems are currentlyregarded as the most suitable systems for large-scale cell culturebecause they have the highest surface to volume ratio and enable bettermonitoring and control. Nevertheless, current microcarrier culturesystems have a number of serious disadvantages: small microcarriercultures cannot be used to inoculate larger microcarrier cultures;therefore, a production facility must use other culture systems for thispurpose; the cost of microcarriers is high, which can necessitatereprocessing of the microcarriers for reuse with the attendant costs;and the oxygen transfer characteristics of existing microcarrier systemsare rather poor.

[0007] Specific forms of calcium phosphate ceramic have been identifiedfor use in microcarriers to support anchorage-dependent cells insuspension. These specialized ceramics provide a material which isbiomimetic, i.e., it is composed of mineral species found in mammaliantissues, and which can be further applied to a variety of in vitrobiological applications of commercial interest. A number of common celllines used in industrial applications require attachment in order topropagate and need substrate materials such as microcarriers for largescale cultivation. U.S. Pat. No. 4,757,017 (Herman Cheung) teaches theuse of solid substrates of mitogenic calcium compounds, such ashydroxylapatite (HA) and tricalcium phosphate (TCP) for use in in vitrocell culture systems for anchorage-dependent mammalian cells. The uniquefeatures of this technology include the growth of cells in layers manycells thick, growth of cells that maintain their phenotype and theability to culture cells for extended periods of time. Cheungdemonstrated the application of this technology for culturing red bloodcells. A current limitation of this technology is that the microcarriersare only available in a non-suspendable granular form. The density ofthese microcarriers further limits the ability to scale-up thistechnology for large bioreactors, which require a suspendable microbeadcarrier.

[0008] A complementary system using an aragonite (CaCO₃) is disclosed inU.S. Pat. No. 5,480,827 (G. Guillemin et al). Although this patent alsoteaches the importance of calcium in a support system for mammalian cellculture, the calcium compound was not in a suspendable form.

[0009] The concept of fabricating a suspendable microcarrier bead with aminor component of glass was discussed by A. Lubiniecki in Large-ScaleMammalian Cell Culture Technology in which a minimal glass coating wasapplied to a polymer bead substrate by a chemical vapor depositionprocess or low temperature process. This approach also was disclosed inU.S. Pat. No. 4,448,884 by T. Henderson (see also U.S. Pat. Nos.4,564,532 and 4,661,407). However, this approach primarily used thepolymer bead substrate to maintain suspendability.

[0010] An example of the use of non-suspendable or porous ceramicparticles for cell culture is taught by U.S. Pat. No. 5,262,320 (G.Stephanopoulos) which describes a packed bed of ceramic particles aroundand through which oxygen and growth media are circulated to encouragegrowth of cells. U.S. Pat. No. 4,987,068 (W. Trosch et al.) also teachesthe use of porous inorganic (glass) spheres in fixed bed or fluidizedbed bioreactors. The pores of the particles act as sites for the cultureof cells. Conversely, Richard Peindhl, in U.S. Pat. No. 5,538,887,describes a smooth surface cell culture apparatus which would limit cellattachment to chemical adhesion and prevent mechanical interlocking.

[0011] Macroporous glass beads also have been reported by D. Looby andJ. Griffiths, “Immobilization of Cells In Porous Carrier Culture”,Trends in Biotechnology, 8: 204-209, 1990, and magnesium aluminateporous systems by Park and Stephanopolous, “Packed Bed Reactor WithPorous Ceramic Beads for Animal Cell Culture, BiotechnologyBioengineering, 41: 25-34, 1993. Fused alumina foams have been reportedby Lee et al, “High Intensity Growth of Adherent Cells On a PorousCeramic Matrix. Production of Biologicals from Animal Cells in Culture,editors, R. E. et al, Butterworth-Heinemann pp. 400-405, 1991, andpolyurethane foam by Matsushita et al, “High Density Culture ofAnchorage Dependent Animal Cells by Polyurethane Foam Packed Bed CultureSystems”, Applied Microbiology Biotechnology, 35: 159-64,1991.

[0012] Fluidized bed reactors have been used for cell culture asreported by J. M. Davis (editor), Basic Cell Culture, (Cartwright andShah), Oxford University Press, New York, 1994, but require carriersystems with densities between 1.3 and 1.6 g/cc. According to Cartwright(J. M. Davis, supra.), generally, in fluidized beds, cells do not growon the exterior surface of carriers where they would be dislodged byinter-particle abrasion. Instead, as with macroporous microcarriers,they colonize the interior pores where they proliferate in a protectedmicroenvironment. As examples, (Cartwright, supra, p. 78) cell carriersused in fluidized beds include glass beads (Siran by Schott Glass), andcollagen microspheres produced by Verax. Cartwright also disclosed otherconventional microcarriers weighted with TiO₂ (Percell Biolyticaproducts) and IAM-carrier polyethylene beads weighted with silica.

SUMMARY OF THE INVENTION

[0013] Examples of the microcarriers of the present invention are setforth in FIG. 1.

[0014] The present invention provides hollow microbeads having a densityof about 1.01 grams/cc to about 1.12 grams/cc. More specifically, thehollow microbeads comprise 0 to 100% hydroxylapatite (HA), 0 to 100%tricalcium phosphate (TCP) and/or 0 to 100% other calcium phosphatecompounds. The hollow microbeads comprise a wall, wherein the wall maybe impermeable to aqueous media. The essentially spherical hollowmicrobeads can have a diameter of from about 100 micrometers to about 6millimeters. In another embodiment, the hollow microbead can furthercomprise a porous coating and/or a biological coating.

[0015] The present invention also provides hollow microbeads having adensity from about 1.2 grams/cc to 2.0 grams/cc. These hollow microbeadscan further comprise a porous coating and/or a biological coating.

[0016] Also provided are biomedical implants comprising theabove-described microbeads. The biomedical implants can further comprisea biological material or pharmaceutical agent. More specifically, thebiomedical implants have a density from about 25% to about 75% of thematerial's theoretical density. (By “theoretical density” is meant thedensity of a microbead having no pores.) The biomedical implant maycomprise a microbead wherein the microbead comprises a wall that isessentially impermeable or porous to aqueous media. The biomedicalimplant may also comprise microbeads comprising holes, i.e., portals orchannels.

[0017] Also provided are chromatographic columns comprising the hollowmicrobeads as set forth above.

[0018] Also provided are aggregates comprising the hollow microbeads asset forth above. Such aggregates can be used as biomedical implants andchromatographic columns. The aggregates may be bonded by cementationsagents.

[0019] The invention further provides hollow and solid glass or polymermicrobeads formed with or coated with particulate HA, TCP and otherCaPs.

[0020] Also provided are hollow and solid microbeads comprisingcomposites of HA, TCP, other CaPs and ceramic, including glass andpolymeric materials. These microbeads may have abraded surfaces andaggregates may be made from them. The aggregates may be used inbiomedical implants and in chromatographic columns.

[0021] Also provided are suspendable and non-suspendable aggregatescomprising closed and/or open pores, foamed structures of ceramic,including glass, and/or polymeric composite materials. These aggregatescan comprise HA, TCP and other CaP coatings, porous coatings, orbiological coatings including growth factors. Biomedical implants can bemade comprising these aggregates. Also provided are methods ofaugmenting tissue comprising implanting these biomedical implants. Thebiomedical implants may further comprise a biologically active agent andhave a density from about 25% to about 75% of the material's theoreticaldensity. Chromatographic columns also can be made from such aggregates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows examples of microcarriers (microbeads) of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] CaP Microbead Processing Method for Producing a SuspendableMicrocarrier Substrate

[0024] The present invention relates to suspendable microbeads (alsoreferred to herein as microcarriers and microspheres). Thesemicrocarriers (FIG. 1) can be used for mammalian cell culturingapplications requiring anchorage-dependent cells in laboratory andcommercial bioreactors. The microspheres may be produced by specializedslurry processing and subsequent use of a coaxial blowing nozzle, tocreate the enclosed porosity structure of the microspheres forsuspension in aqueous-based cell culturing media. More specifically, theCaP microsphere is hollow and comprised of suitable mixtures of 0 to100% HA, and 0 to 100% TCP, and/or other calcium phosphate compoundsincluding any mixture thereof. The preferred process for the CaPmicrocarrier ceramic slurry process uses a nozzle-reactor system withslurry droplet blowing agent, to produce hollow microspheres in a sizerange of about 0.5 millimeter to about 6 millimeter diameter andspecific wall thicknesses that sinter to an aqueous impermeable statesufficient to maintain suspendability in culture medium. As examples,the microcarrier wall thicknesses are about 20 to about 40 micrometersfor 0.5 mm diameter, about 50 to about 80 micrometers for 1.0 mmdiameter, and about 170 to about 230 micrometers for 3.0 mm diametermicrocarriers to maintain suspendability. More specifically, the densityof the microspheres is preferably in the range from about 1.01 gms/cc toabout 1.12 gms/cc. Preferably, the density range is controlled fromabout 1.01 gms/cc to about 1.06 gms/cc. Final hollow microspheredimensions are adjusted to compensate for shrinkage normally encounteredduring sintering from the formed state, which in the case of CaPmixtures of HA and TCP is typically in the range of about 15-25% linearshrinkage.

[0025] Furthermore, using variations in processing conditions or inpost-microsphere fabrication processing, the hollow microspheres canhave either rough (e.g., textured or abraded) or smooth surfaces fortailoring the surface condition to either enhance the attachment ofcells for increasing the production of cell by-products, or to enhancethe release of grown cells in culture for applications requiring theproliferation of cells. The surfaces of the hollow microspheres may alsocomprise other substances, including, for example, biological coatings,including growth factors, selected proteins, amino acids, collagen, andother such materials.

[0026] Additionally, the density of the hollow microsphere can beadjusted to about 1.2 gms/cc to about 2.0 gms/cc for fluidized or fixedbed applications. The microsphere wall thickness may be increased forany given suspendable size to achieve this range. Also, for fixed bedapplications, the density of the microcarrier may be greater than 1.6gms/cc.

[0027] Alternate Method for Fabricating Hollow CaP Microcarriers withDiameters Less Than 500 Micrometers (Sol Gel Type System)

[0028] In this aspect of the invention, a reaction precipitation methodfor producing submicron CaP microspheres (FIG. 1, 1.2) is used inconjunction with an oleyl alcohol condensing solution to fabricatehollow microspheres. The CaP precipitated solution is nozzle-sprayedonto the oleyl alcohol using nozzle size, pressure, and spray distanceto control the size of the microsphere. This method utilizes a lowsolids to liquid ratio (typically less than about 20%) in conjunctionwith hot oleyl alcohol for concentrating the CaP solids into a shell forforming the microspheres. After the fabricating step, the microspheresare dried, sintered and classified to size and density by traditionalsieving and air classification methods. The microspheres also may beclassified to desired densities for buoyancy by liquid densityseparation methods. The degree of sintering also is used to control theporosity or permeability of the microsphere. Therefore, a suspendable ornon-suspendable (porous/non-porous) microcarrier may be produced by thismethod. More specifically, for fixed bed or fluidized bed reactors,higher density microcarriers are produced to meet the requirements forthese non-suspendable applications.

[0029] Alternate Method for Fabricating Hollow CaP Microspheres byCoating on Wax or Other Organic Microbeads (FIG. 1, 1.2)

[0030] Slurries or powders of calcium phosphate compounds are applied tothe surfaces of wax or other organic microbeads. The organic microbeadis removed by thermal decomposition, solvent extraction, or acombination thereof. The slurries or powders may incorporate binder toincrease the strength of the shell formed on the wax or organicmicrobead, and may further aid in maintaining the formed CaP microsphereduring removal of the wax or organic substrate. If required, the slurrysolids content can be adjusted to increase or decrease the density ofthe CaP microsphere. Likewise, powder can be compacted around the wax ororganic microbead to increase the density of the resulting CaPmicrosphere. After the fabrication step, the microspheres are dried,sintered and classified to size and density by traditional sieving andair classification methods. The microspheres also may be classified todesired densities for buoyancy by liquid density separation methods. Thedegree of sintering also is used to control the porosity or permeabilityof the microsphere. Therefore, a suspendable or non-suspendable(porous/non-porous) microcarrier may be produced by this method. Morespecifically, for fixed bed or fluidized bed reactors, higher densitymicrocarriers are produced to meet the requirements for thesenon-suspendable applications.

[0031] Porous CaP Coatings for Bonding to the Substrate Surfaces ofHollow or Solid Beads or Microbeads Comprised of CaP, Glass, Other OxideCeramics or Polymers, Proteinaceous Materials or Composite Materials(FIG. 1, 1.1 and 1.3)

[0032] A further object of this invention relates to porous CaP coatingsthat are comprised of suitable mixtures of particulate HA, TCP and/orother calcium phosphate compounds, and varying amounts of porosity forcell culturing applications. The benefits of CaP coatings includeenhanced cell attachment, the protection of cells during culturing, andincreased surface area for cell proliferation on a variety ofsubstrates, including dense CaP and other dense or porous substratematerials. The purpose of CaP coatings on non-CaP substrates is toenhance the beneficial bioactivity of the substrate surface in cellculturing applications.

[0033] More specifically, two size ranges of porosity have beenidentified which include pore sizes less than about 30 micrometers forincreased chemical activity of the substrate, and about 30-80micrometers for in-growth by cells, protection of cells and enhancedchemical activity of the substrate. For either pore size range, theamount of porosity is typically greater than about 10% but less thanabout 60% to ensure mechanical integrity of the coating. Morespecifically, the amount of porosity to ensure interconnectivity shouldbe greater than about 20%. Appropriate methods for applying CaP coatingsinclude a slurry coating technique and/or applying adherentpowder/particulates. The coatings may comprise open and/or closedporosity. Specific “pore formers” which produce the desired pore sizerange include various organic materials, which are varied in amount toproduce porous CaP coatings for use in cell culturing applications.During the thermal processing cycle for sintering, the pore formersdecompose leaving channels of interconnected porosity for pore volumestypically greater than about 20%.

[0034] Microbead substrate materials to be coated include, for example,dense CaP, and other oxide ceramics such as alumina, mullite, porcelainor glass. These substrate materials may be used to produce hollow orsolid microspheres for cell culturing applications. The CaP coatings areapplied to either unsintered or pre-sintered microspheres, andsubsequently re-sintered for bonding. The substrate also may be reheatedto soften the surface in the case of a glass substrate. The CaP coatingcan be applied to porous substrates and then co-sintered to enhancebonding and further densify the substrate layer. For polymeric andproteinaceous substrates, the preferred method for bonding is reheatingthe substrate material to soften the surface or applying a secondarybio-adhesive material to provide a bonding layer.

[0035] Aggregate Suspendable Microcarriers with Open Porosity (FIG. 1,1.4)

[0036] CaP microcarrier spheres are prepared as described above using anozzle method from either a modified sol gel process or powder slurryprocess. The preferred embodiment of this aspect of the inventionrequires hollow microspheres with densities less than 1 gm/cc which aresubsequently bonded with CaP-prepared powder slurry, calciumphosphate/sulfate cements, or sol gel-modified slurries in the unfiredstate for producing aggregates. Lower initial microsphere densities arerequired to offset the additional weight of the bonding slurries forachieving final suspendability in the range of about 1.00 gms/cc toabout 1.12 gms/cc. After sintering, aggregates are sized by well knownceramic granulating/grinding and sieving methods. The sized aggregatesare then sorted for density by liquid density separation methods. Liquiddensities are prepared in the desired range for buoyancy and aggregatesare separated based on the suspending properties. The starting spheresize can be used to control the open pore size and pore sizedistribution. One advantage of this method is that the open pores areused as sites for cell attachment and growth, thereby providingprotection to the cultured cells and additional surface area forenhancing growth of cells. As stated above, the aggregate density isincreased by addition of bonding slurry and/or additionally increasingthe wall thickness of starting microspheres for use in fixed bed orfluidized bed reactors as examples of non-suspendable applications.

[0037] Aggregate Suspendable Microcarriers with Closed Porosity (FIG. 1,1.5)

[0038] As an alternative to a large, single void hollow microsphere, theclosed porosity of the microcarrier can be distributed in multiple,isolated pores to reduce the density of the CaP in meeting thesuspendability requirement. Closed porosity refers to microcarrierswherein voids are separated by dense material not open to the externalsurface. The preferred approach for creating closed porosity within theaggregate consists of the use of a closed-cell organic pore formermaterial which produces about 60% to 70% closed porosity within theaggregate. After sintering, aggregates are sized by well known ceramicgranulating/grinding and sieving methods. The sized aggregates may thenbe sorted for density by liquid density separation methods. Liquiddensities may be prepared in the desired range for suspendability andaggregates are separated based on the suspending property. As statedabove, the aggregate density is increased for use in fixed bed orfluidized bed reactors.

[0039] Composite Suspendable or Non-Suspendable Microcarriers (FIG. 1,1.0 and 1.6)

[0040] The preferred embodiment of this aspect of the invention relatesto the incorporation of substantial volume fraction of CaP powder orparticulate filler in a hollow or solid natural or synthetic polymer(e.g., polyethylene, polystyrene, dextran, gelatin) and/or glassmicrosphere. More specifically, the void volume in the hollowmicrosphere structure of a polymer or glass reduces the bulk density. Asa result, the microcarrier can accommodate a higher CaP filler solidscontent and still meet the suspendability requirement. Thesemicrocarriers employ a variety of hollow glass or polymer microsphereprocessing methods. Subsequently, a CaP filler is incorporated toincrease density to the desired suspendability.

[0041] As an alternative to the hollow polymer or glass microsphere,substantial amounts of “closed porosity” in the polymer or glass alsoallows for higher CaP filler additions due to the lower initial bulkdensity of the polymer or glass component in the composite. The closedporosity in the polymer or glass can be created, for example, by afoaming agent or control of sintering parameters to produce closedporosity. After the composite microsphere having a size of about 100 toabout 500 micrometers is made, the surface can be modified by abradingto increase exposure of the CaP filler for enhanced cell attachment andgrowth activity. The density of the microcarrier composite also can beincreased using higher levels of CaP filler in either a hollowmicrosphere or solid form for fixed bed or fluidized bed reactorapplications. Additionally, non-suspendable or suspendable microcarriersmay be fabricated from monolithic (continuous solid) forms of combinedCaP filler and polymer and/or glass materials (without or with porosity)which are subsequently granulated, ground or chopped into desired sizesand shapes.

[0042] CaP Non-Suspendable Microcarrier with Open Porosity (FIGS. 1,1.2, 1.7 and 1.8)

[0043] CaP non-suspendable microcarrier spheres with open porosity areprepared as described above using a conventional spray drying method orpelletizing method well known in the art from either a modified sol gelprocess or powder slurry process. Also the method taught by Martin (U.S.Pat. No. 3,875,273, supra.) can be used to form open porousmicrocarriers. The preferred shape of the individual microcarrierproduced by the methods described above is sphere-like with a continuousporous phase (FIGS. 1, 1.8). An alternative shape of microcarrier is ahollow microsphere having a continuous porous wall that connects thecentral microsphere void to the outer surface (FIGS. 1, 1.2). This formof microcarrier can be produced by the reactor nozzle method taught byTorobin (U.S. Pat. No. 5,397,759). The open porosity of microspheres iscreated by sintering at a lower temperature of about 1100° C., which isless than that typically required to densify the material, or by addinga pore former as previously discussed, followed by sintering. Thepreferred embodiment of this invention requires microspheres withdensities greater than 1.12 gm/cc for packed or fluidized bedbioreactors.

[0044] The above-described microspheres can be subsequently bonded withCaP-prepared powder slurry or sol gel-modified slurries in the unfiredstate for producing aggregates. The aggregate form is created bysintering the agglomerates in a fabricated form. After sintering,aggregates are sized by well known ceramic granulating/grinding andsieving methods. An alternative bonding method comprises the use of CaPor calcium sulfate cement or other cement for bonding microspheres. Oneadvantage of the aggregate method for fluidized bed applications is thatthe open pores of the bonded aggregate are used as sites for cellattachment and growth, thereby providing protection to the culturedcells and additional surface area for enhancing growth of cells. Thestarting sphere size can be used to control the open pore size and poresize distribution for optimizing cell growth of different cell types.The density of these microcarriers will, in general, be greater than1.12 gms/cc in either individual microcarrier or aggregate form.

[0045] CaP Microspheres for Chromatographic Applications

[0046] The processing methods as set forth for CaP microspherefabrication (e.g., CaP Microbead Processing Method for Producing aSuspendable Microcarrier Substrate and Alternate Method for FabricatingHollow CaP Microcarriers with Diameters Less Than 500 Micrometers (SolGel Type System) described above) can be used to produce microspheresfor use in chromatographic applications. In this application, apreferred embodiment for the microspheres is in the size range of about10 to 100 micrometers diameter with open porosity in the range of about20% to about 60% and a pore size range from about 0.01 to about 0.5micrometers. Microsphere sizes larger than 100 micrometers, up to about2 millimeters diameter in either the hollow or non-hollow sphere form,improve permeability by minimizing the resistance to flow in thechromatographic column while maintaining the ability to separate andpurify proteins, enzymes, nucleic acids, viruses, and othermacromolecules. In addition, the thin wall of the hollow porousmicrosphere improves permeability for greater efficiency of separationand purification.

[0047] Implantable CaP Microspheres and Aggregates of BondedMicrospheres

[0048] The processing methods as set forth for CaP microspherefabrication (e.g., CaP Microbead Processing Method for Producing aSuspendable Microcarrier Substrate and Alternate Method for FabricatingHollow CaP Microcarriers with Diameters Less Than 500 Micrometers (SolGel Type System) described above) also can be used to producemicrospheres for use as a biomedical implant. In this application, apreferred embodiment for the microspheres is in the size range of about500 micrometers to about 1,000 micrometers diameter. Compacts ofmicrospheres in this size range produce an interstitial open porosity ofabout 60% with a pore size range of about 350 micrometers to about 500micrometers. For other tissue in-growth applications, such as epithelialtissue, the microcarrier diameter size range can be adjusted to providean open pore size range from about 50 micrometers to about 150micrometers to facilitate tissue in-growth.

[0049] As stated previously, aggregates can be formed by bondingmicrospheres using the CaP slurry and cement methods (e.g., CaPNon-Suspendable Microcarrier with Open Porosity) and can be used forbiomedical implant applications. Cemented aggregates offer the advantageof conformation to the implant site without subsequent sintering. Inaddition, these microspheres can be used with collagen to form acomposite implantable material. The open pore size within themicrosphere-bonded aggregates can be adjusted for specific tissuein-growth as stated above.

[0050] The microspheres and aggregates of microspheres discussed abovecan be used as carriers of biological growth factors and otherpharmaceutical agents including anti-inflammatory and anti-tumor agents.Open porosity within the microsphere can be made by sintering at atemperature less than that required to fully densify the material, or byadding a pore former to the material. This open porosity can be adjustedto facilitate delivery of specific biological growth factors andpharmaceutical agents. The tissue growth factors or pharmaceuticalagents are incorporated either as a coating on microspheres oraggregate, or are impregnated within the open porosity of themicrosphere or aggregate. The size of the open porosity betweenindividual microspheres and within aggregates of microspheres can beadjusted by changing the size of the microspheres. Also, thesemicrospheres and aggregates can be used to culture tissues which may besubsequently implanted to augment tissue defects.

[0051] Hollow microspheres and hollow microspheres bonded in aggregatesprovide a central cavity as a reservoir for growth factors or otherpharmaceutical agents. The hollow microsphere provides compressivestrength due to its geometry as a basic sphere in distributingmechanical stress within its wall. In addition, the thin wall of thehollow microsphere can be replaced by tissue in-growth as the materialwithin the wall resorbs. The degree of resorbability may be adjusted bychanging the wall thickness, the amount and size of porosity within thewall, and the amount of HA, TCP, and/or other CaP phases. Generally, anincreased amount of TCP increases the resorbability of the microsphere.

[0052] Aggregates of the material can be shaped by hand carving ormechanized grinding. The degree of carvability can be adjusted bychanging the strength of the bond between the microspheres. This bondstrength can be changed by adjusting the sintering temperature of thebonding slurry or by adjusting the bonding cement chemistry. Aspreviously stated, aggregates formed with cements can also be molded andallowed to set and conform to the implant site, thereby reducing theneed for grinding or carving to shape.

[0053] A typical application for microspheres and bonded aggregates ofmicrospheres is the repair and augmentation of bony defects. Also,microspheres of smaller sizes can be used to augment soft tissue, e.g.,cartilage defects.

[0054] An object of this invention is to provide appropriate forms ofcalcium phosphate ceramic materials to be fabricated in specific shapesand sizes for anchorage-dependent mammalian cell culturing applications.Fabricated forms of the calcium phosphate ceramic to be used asmicrocarriers in, for example, mammalian cell culture applicationsinclude hollow microspheres, solid spheres, aggregates of microspheres,multi-pore aggregates, polymeric and glass CaP composites. A variety ofcoatings that can be made highly porous or combined with organic orpolymeric materials, including growth factors, to form compositestructures also can be used in conjunction with these fabricated forms.Combinations of the aforementioned fabricated forms also can be used tomake the microcarriers of the present invention. To achieve the objectsof this invention, appropriate mixtures of hydroxylapatite, tricalciumphosphate, and/or other CaP compounds and, in certain cases, an openpore phase are used to enhance cellular growth through the highersurface area of the porous structure. Closed porosity is used tomaintain buoyancy in growth media. Although more limited in application,calcium carbonate can be used in granular form, as a coating on asubstrate carrier for cell culturing applications or as the Ca phase inpolymeric composites. A major advantage of the CaP ceramic microcarriersis that the finished material can be heated to as high as 1,000° C. fordecomposing organic cell culture components to recycle the microcarrierafter its use in culture. This heating step can not be done with thepolymeric/CaP composite microcarriers described herein. Other advantagesof the CaP substrate as a microcarrier for cell culturing applications,as compared to polymeric materials, are that the CaP substrate isdimensionally stable, due to non-swelling, by absorption of media.

[0055] For bioreactor applications which require a suspendable form ofmicrocarrier, the preferred form of the calcium phosphate substratecomprises a hollow microcarrier of about 0.2 to about 6 mm diameter thathas been sintered to a sufficiently dense, impervious or impermeablestate. For anchorage-dependent mammalian cells, the hollow microcarrieris suspended in the growth medium of the bioreactor. Therefore, a degreeof buoyancy for the microcarrier is required. The preferred microcarrierdensity is in the range of about 1.00 to about 1.12 gms/cc. The densityis more preferably in the range of about 1.00 to about 1.06 gms/cc. Thehollow microspheres are fabricated from a CaP ceramic based on mixturesof HA and TCP, and/or other CaP compounds. The microsphere wall is of acontrolled thickness in the unsintered state that varies depending onsphere size (see Table 1). The microsphere wall is sintered to asufficiently dense state of about 3.0 gms/cc, or greater for HA/TCPmixtures to maintain an impervious state for suspendability.Alternatively, the open porosity structure can be sealed with apolymeric or other organic film former to achieve an impervious state.

[0056] U.S. Pat. No. 5,397,759 by Leonard Torobin teaches a process forfabricating porous ceramic hollow microspheres of uniform diameter anduniform wall thickness in sizes ranging from 1-4 mm in diameter. U.S.Pat. No. 5,225,123 by Leonard Torobin also teaches a process forfabricating hollow ceramic microspheres with closed porosity. Thepresent invention uses aspects of this technology to produce asuspendabie calcium phosphate ceramic for use in cell cultureapplications. An alternative technology for fabricating porous ceramichollow spheres is described in U.S. Pat. No. 3,875,273 by Robert M.Martin. There also are other processes for fabricating hollowmicrospheres from ceramic materials as described by David Wilcox inHollow and Solid Spheres and Microspheres: Science and TechnologyAssociated With Their Fabrication and Application, Materials ResearchSociety Symposium Proceedings, Volume 372, 1995. These fabricationmethods include sacrificial cores, nozzle-reactor systems,emulsion/phase separation techniques (including sol gel processing), andmechanical attrition. Although these approaches do not specificallyaddress calcium phosphate materials or cell culture microcarrier systemapplications, the described processes could be modified to produce themedia suspendable calcium phosphate microcarrier systems of the presentinvention based on teachings herein. However, the aforementionedtechnologies must be combined with either commercial sources of calciumphosphate materials or be allied to the chemical formulation ofhydroxylapatite and/or other calcium phosphate compounds such astricalcium phosphate (tribasic calcium phosphate) in order to producethe carriers of the present invention. These materials are of primaryinterest, since they can be fabricated in dense non-permeable forms astaught by A. Tampieri in the Journal of Material Science: Materials inMedicine, Vol. 8, pp. 29-37, 1997.

[0057] The efficacy of cell culture microcarriers foranchorage-dependent cells can also be greatly improved by the use ofcoatings that enhance cell attachment. Cartwright and Shah in Basic CellCulture (J. M Davis, supra.) indicate collagen, fibronectin, laminin,and Pronectin (synthetic fibronectin promoting better attachment) arecoatings currently used to promote cell attachment. Cheung (Cheung,supra.) and others have also reported poly-lysine as a coating thatpromotes cell attachment and proliferation.

[0058] Other applications of hydroxylapatite bead materials(non-suspendable) for biotechnology include, for example, use forchromatographic filtration/separation columns as taught by Louis Langein U.S. Pat. No. 5,492,822 for the isolation of human pancreaticcholesterol. The HA form of CaP is also used for the separation andpurification of proteins, enzymes, nucleic acids, viruses, and othermacromolecules. According to Bio-Rad in Bulletin No. 1115, HA has uniqueseparation properties and high selectivity and resolution. Applicationsof HA chromatography include separation of monoclonal and polyclonalantibodies of different classes, antibodies which differ in light chaincomposition, antibody fragments, isozyme, supercoiled DNA from linearduplexes, and single-stranded from double-stranded DNA.

[0059] For biomedical implant applications, HA has been used inparticulate, monolithic and coating forms for tissue augmentation,primarily bone. HA has a number of advantages as an implant materialincluding biological attachment to bone tissue, outstandingbiocompatibility, elastic modulus close to that of bone, otherreasonable mechanical properties such as strength, and support of newbone growth.

[0060] HA also may be used as a host material for delivery of biologicalmaterials, including growth factors, and referred to herein asbiological coatings, and other pharmaceutical agents includinganti-inflammatory and anti-tumor agents. Hideki Aoki in MedicalApplications of Hydroxyapatite discusses the properties and uses of HAfor biomedical applications. U.S. Pat. No. 5,422,340 teaches the use ofcalcium phosphate particles as carriers for bone growth factors. Forbone in-growth applications of HA, Kenna in U.S. Pat. No. 5,192,324teaches that a particle size of +30 to −20 U.S. Standard mesh sizeprovides pore sizes of 350 micrometers to 500 micrometers to facilitatebone in-growth.

[0061] Hydroxylapatite can be produced by a variety of methodsincluding: 1) preparation from calcium nitrate and ammonium phosphate(E. Hayek and H. Newesely in Inorganic Synthesis, Jacob Kleinberg,editor, McGraw-Hill, New York, Vol. 7, pp 63-65, 1963, or S. Larsen etal in Experimentia, Vol. 27, No. 40, pp. 483-485, 1971, 2) synthesisfrom calcium hydroxide and phosphoric acid as described by J. Tagai etal in Adv. Biomaterials 2, pp 477-488, 1980, 3) sol gel processing asdescribed by A. Deptula in the Journal of Non Crystalline Solids 2, pp.477-488, 1980, and 4) hydrolysis of CaHPO₄ under heat and pressure(hydrothermal bomb) as described by A. Posner et al, Acta Cryst., 11, p308, 1958. Likewise, variations of these methods, well know in the art,are also used to produce hydroxylapatite.

[0062] Tricalcium phosphate can be produced by the partial decompositionof hydroxylapatite upon calcining at >1200° C. (A. Tampieri, supra.)and/or the reaction of a stoichiometric mixture of hydroxylapatite withCaHPO₄ with subsequent thermal processing at or above 900° C. asdescribed by C. Rey (Biomaterials 11, p. 13 (1990).

EXAMPLE 1 Process for Producing CaP Hollow Microspheres for MammalianCell Culturing Applications

[0063] CaP hollow ceramic spheres in the range of about 0.2 mm to about6 mm in the unsintered state are produced from ceramic slurries usingeither a commercial source of starting CaP raw material (calciumphosphate tribasic) or a precipitated form of CaP based on the nitratesolution process as taught by Jacob Kleinberg, editor, InorganicSynthesis, McGraw-Hill, New York, Vol. 7, pp. 63-65 (Hayek andNewesley), 1963. Using the commercial source of CaP raw material (FMC,Lawrence, Kans.) slurry processing properties are controlled byoptimizing several variables, including slurry density-related to solidscontent, viscosity, particle size distribution, film stabilizing agents,and dispersing agents. The characteristic spheres are formed fromslurries at around several thousand per minute. After forming thespherical geometry from the nozzle, the spheres are subsequently driedin free flight by loss of solvent with the aid of a high vapor pressureorganic as the dispersing liquid. The dried spheres in the green stateare polymer-bonded shells which are then conventionally fired to sinterthe walls.

[0064] In more detail, the ceramic slurry is fed through the outerorifice of the nozzle, and the pressurized forming gas that produces themicrosphere void is fed through a center orifice with the outer orificeacting as a metering area for size control. The basic coaxial nozzleprocess is set forth in U.S. Pat. Nos. 5,225,123 and 5,397,759 byTorobin. The forming gas, described above, acts as a blowing agentwithin the slurry droplet and expands during flight from the drop tower.This method of hollow microsphere formation is set forth in Wilcox,Hollow and Solid Spheres and Microspheres: Science and TechnologyAssociated With Their Fabrication and Application, Materials ResearchSociety Symposium Proceedings, Volume 372, 1995.

[0065] The advantage of the nozzle-reactor system is that it has beendemonstrated, for a variety of ceramic materials, to meet exactingspecifications on sphericity, size and wall thickness control.Well-designed nozzle systems dispense individual CaP slurry droplets,with precise control of dimensions, into a reactor for forming themicrosphere geometry. In the reactor, the slurry droplet is converted toa firm hollow microsphere. The reactor in drop tower configuration isthe preferred method for fabricating CaP hollow microspheres for use assuspendable microcarriers for anchorage-dependent cells grown incultures.

[0066] Sintering of the fabricated hollow microspheres is conducted inthe temperature range of about 1100° C. to 1350° C., for time periods ofabout 0.1 to 6 hours to densify the outer wall of the microsphere to asufficient level of impermeability to ensure the suspendability requiredin typical bioreactor applications using aqueous media. The product thatresults from this process comprises hollow microspheres with thepreferred sphere densities of about 1.00 to about 1.06 gms./cc. Fortypical sphere diameters in the range of about 1 to 3 millimeters,sphere wall thicknesses must be controlled to size limits within therange of about 75 to about 250 micrometers to achieve sphere densitiesslightly greater than 1.00 gms/cc for optimizing suspendability. Table 1sets forth the relationship of sphere density as a function of spherewall thickness for typical 0.5 mm, 1 mm, 2 mm, and 3 mm diametermicrospheres based on a typical calcium phosphate density of 3.0gms./cc. TABLE 1 CaP Sphere Diameter, Wall Thickness, Sphere DensityTables Based on CaP Density of 3.0 Grams Per Cubic Centimeter SphereDia. (mm) Wall Thk. (microns) Sphere Density (gms/cc) 0.5  25 0.81 0.5 30 0.96 0.5  35 1.09 0.5  40 1.22 0.5  50 1.46 0.5  65 1.78 0.5  751.97 0.5 100 2.35 1.0  25 0.43 1.0  50 0.81 1.0  60 0.96 1.0  65 1.021.0  75 1.16 1.0 100 1.46 1.0 125 1.73 2.0  75 0.63 2.0 100 0.81 2.0 1250.99 2.0 150 1.16 2.0 175 1.32 2.0 200 1.46 2.0 225 1.60 2.0 250 1.733.0 100 0.56 3.0 125 0.69 3.0 150 0.81 3.0 175 0.93 3.0 190 1.00 3.0 2001.05 3.0 210 1.09 3.0 225 1.16 3.0 250 1.26

[0067] An alternative method for fabricating ceramic hollow spheres isset forth in U.S. Pat. No. 3,875,273 by Martin and can be used tomanufacture the CaP microspheres of the present invention. However, itis not as readily scaled for high volume production needs. The Martinmethod produces porous ceramic microbeads which subsequently requiresealing of the outer microsphere surface with a polymeric film to attaina suspendable microcarrier.

EXAMPLE 1A Alternative Method for Producing CaP Hollow Microspheres withDiameters in the Size Range From 0.2 mm to 6.0 mm by Coating Wax orOther Organic Microbeads with CaP Compounds

[0068] Hollow ceramic microspheres may also be fabricated by coatingceramic powders or slurries onto microbeads of wax or other organicmaterials and subsequently removing the wax microbead through thermaldecomposition and/or solvent extraction. The resulting hollow ceramicmicrospheres are sintered to the desired density.

[0069] More specifically, a microbead of polyethylene wax or other waxor organic material is formed by spraying from a melt and re-solidifyingat a lower temperature. Size of the microbeads is determined by the sizeof the spraying orifice and the pressure under which the organicmaterial or wax is sprayed. Wax or other organic microbeads also can beproduced, for example, by compaction of wax powders by rolling in heatedball mills or pan pelletizers or by rolling the powders and graduallyadding a solvent to the powders to consolidate them in the form ofbeads. The size of the beads is controlled by the particle size of thestarting powder, heat of the ball mill or pan pelletizer, speed ofrotation of the ball mill or pan pelletizer, size of the ball mill orpan pelletizer, length of rolling time, and amount and speed of additionof an organic solvent system. The desired size of bead is obtained byscreening. This screening process also removes the unconsolidatedpowders from the powder consolidation method.

[0070] In the case of hollow ceramic (CaP) microspheres prepared fromslurries, powders with a broad particle size distribution are preparedby ball milling dry powders. These powders are further reduced inparticle size by wet milling. By this means, a slurry with a high solidscontent can be prepared as is well known in the art of preparation ofceramic powders and materials. Likewise, dispersants and binders can bemilled with the slurry to produce a higher solids content, promoteadherence to the wax/organic microbead and to promote stronger resultingceramic microspheres. The solids content of the slurry controls thefinal density/porosity of fabricated ceramic microbead in the unsinteredstate. The prepared slurry is mixed with the previously described wax ororganic microbeads of the desired size. The mixture is sprayed underpressure through an orifice of sufficient size to allow passage of thewax/organic beads with a coating of slurry. The slurry coating of thewax/organic microbeads may also be accomplished by converging the slurrymixture with a liquid mixture of the wax/organic microbeads such thatthe two streams of materials converge causing the coating of the ceramicslurry onto the wax/organic microbeads. The slurry-coated wax/organicmicrobeads are allowed to dry during falling in air or by drying inheated air sufficient to cause drying of the coating, but not melting ordecomposing of the wax/organic microbead.

[0071] The coated microbeads are further classified to the desired sizesby screening through screens of the desired mesh sizes. The wax isremoved from the ceramic-coated wax beads by heating the coated beads tomelt and decompose the wax/organic substrate. The porosity of theunsintered ceramic-coated shell allows for the removal of thewax/organic substrate by melting and thermal decomposition. Likewise,the porosity of the unsintered ceramic-coated shell allows for removalof the wax/organic by solvent extraction or a combination of solventextraction and/or thermal decomposition. After the fabricating step, themicrospheres are further dried, sintered and classified to size anddensity by traditional sieving and air classification methods. Themicrospheres also may be classified to desired densities for buoyancy byliquid density separation methods.

[0072] In the case of preparation by compaction of ceramic powders ontowax/organic beads, wax/organic beads are prepared as previouslydescribed in this example. A fine ceramic powder distribution isobtained by numerous methods well known in the art. An example of such amethod is dry ball milling and subsequent wet ball milling. The wetmilled powder is subsequently dried and further ball milled or air jetmilled to break up agglomerates. The resulting powder and wax/organicmicrobeads of the desired size are placed in a ball mill, pan pelletizeror other container and rolled or vibrated to compact the powders ontothe wax/organic microbead. The use of a dense micro-media may also beadded to a ball mill or other container to further compact the powdersonto the wax/organic microbeads. Furthermore, the resulting shellthickness and density of the ceramic coating is controlled by the energyimparted to the fabricated bead. The amount of energy is controlled bythe amount of time of compaction, and speed of rotation or vibration,and/or addition of liquid to promote the agglomeration of powders ontothe wax/organic microbeads. Excess or unconsolidated powders are removedfrom the coated microbeads by sieving through screens of sufficient sizeto retain the coated microbeads and allow excess powders and compactingmedia to pass through. The wax/organic is removed as previouslydescribed and the ceramic microspheres are classified to size by methodspreviously described in this example, and sintered to the desireddensity. The above-mentioned methods are applicable to the formation ofCaP-coated wax/organic microbeads.

EXAMPLE 2 Method for Fabricating Hollow CaP Microcarriers with DiametersLess Than 500 Micrometers (Modified Sol Gel Type System)

[0073] Using the method set forth by Hayek and Newesley in JacobKleinberg, editor, Inorganic Synthesis, McGraw-Hill, New York, Vol. 7,pp. 63-65, 1963 for the synthesis of hydroxylapatite, basic solutions ofcalcium nitrate and ammonium phosphate are prepared and adjusted to a pHabove 9 to promote the precipitation of hydroxylapatite upon addition ofthe ammonium phosphate to the calcium nitrate. Upon completion of thereaction, the solids are allowed to partially settle and are thendecanted from the reacting solution. The decanting process is thenrepeated three times with distilled H₂O. Care must be taken not to washaway the hydroxylapatite precipitate. Based upon initial reactioncalculations, the precipitate is diluted to a solid concentration ofapproximately 15-20%. A dispersant such as Pluronic (BASF, Parsippany,N.J.) may be titrated into this precipitate until a fluid “water-like”consistency is obtained. Using a method as set forth by Kyung Moh fornon-CaP ceramics (“Sol Gel Derived Ceramic Bubbles”, Hollow and SolidMicrospheres, Material Research Society Proceedings, #372, ed. by D. L.Wilcox, 1995), a solids content consisting of 100 grams of CaP, 16 gramsof acetone and 0.5 grams of methyl cellulose (Dow Chemical, Midland,Mich.) is added and stirred into an even consistency. The mix is coveredwith Parafilm wax (VWR Scientific, Chicago, Ill.) to preventevaporation. This solution is subsequently sprayed into hot oleylalcohol (at 95° C.). The nozzle size, pressure, and distance from theoleyl alcohol can be adjusted to give the desired microsphere size. Themixture of droplets and oleyl alcohol is stirred for approximately 20minutes. The gelled bubbles are subsequently filtered from the oleylalcohol and placed on a refractory dish or plate, dried at ˜100° C. for1 hour and fired at a rate of 100° C./hr to 1100-1300° C. forapproximately 1 hour to obtain dense or semi-permeable hollowmicrospheres depending on the desired permeability. The microspheres canbe separated into desired classifications by well known sieving, airclassification methods, and/or buoyancy classification in solutions ofdesired densities.

EXAMPLE 3 Porous CaP Coatings for Bonding to the Substrate Surfaces ofHollow or Solid Beads or Microbead Materials Comprised of CaP, Glass,Other Oxide Ceramics, Polymers, Proteinaceous Materials or CompositeMaterials

[0074] The surface of the hollow microsphere can also be altered byapplying a porous layer of suitable particulate calcium phosphateceramic which (1) will increase the chemical activity of the materialdue to the higher surface area of the material and (2) through largerinterconnecting pore sizes, can also provide porous channels toaccommodate cell and tissue in-growth.

[0075] The porous calcium phosphate coating composition is comprised ofa commercial source of tricalcium phosphate powder (FMC, Lawrence,Kans.) which is either used as-received, or preferably, is calcined inthe temperature range of 1100° C. to 1250° C. to slightly coarsen thematerial for making porous coatings. The tribasic calcium phosphateceramic powder is readily processed in an aqueous medium or othersolvent if desired. The aqueous vehicle can be used in a process thatreadily accommodates a dispersant for increasing solids while minimizingshrinkage during drying. It also accommodates an organic-based poreformer that is the preferred method for generating porosity within thecalcium phosphate structure after sintering. Typically, the pore formeris added in sufficient quantity (>30 volume percent) to create acontinuous porous phase when higher levels of porosity in the coatingare desired. The aqueous tribasic calcium phosphate slurry is cast ontothe surface of the microcarrier substrate and allowed to dry. Theinclusion of a small amount (˜1%) of an acrylic emulsion binder aids inproviding higher green strength to the unfired microcarrier for improvedhandling, and offers resistance to cracking during the drying process.

[0076] For calcium phosphate hollow microspheres and other solid formsof calcium phosphate to ultimately be sintered to an impervious state,the preferred method for coating the microbead or other substratesurface is to pre-sinter the microbead to a temperature of about1100-1300° C. This provides sufficiently high strength for slurrycoating such that the coating and microcarrier can be co-sintered tobond the coating to the microcarrier substrate. The microcarriersubstrate is densified during final sintering. Final sintering can bedone at a higher temperature typically in the range of 1150-1400° C.This sinters the calcium phosphate microcarrier wall with the coatinghaving residual porosity derived from the pore formers that areincorporated into the coating formulation. The resulting material is atleast a two-layer calcium phosphate structure (the substrate is one ofthe layers) with a dense wall supporting a tailored porous coating layerfor enhancing the chemical activity and in-growth potential for cellsand tissues grown in culture. The amount and size of porosity can bealtered based on changes in coating formulation. The amount and type ofpore former is the primary material variable that controls the porousphase. Typically, the amount of porosity in the coating will be in therange of about 20-60% with the pore size controlled in either a finedistribution or coarser distribution, depending on the application. Thefine pore size distribution is comprised of a majority of pores lessthan about 30 micrometers. The coarser pore size distribution will havepore channels in about the 30 to 80 micrometer range, but the pore sizedistribution can be further altered to produce an even coarser and/orfiner pore size distribution for certain applications.

[0077] As an alternative to the slurry method of coating, a powder orparticulate agglomerate of CaP may be directly bonded to the surface ofthe solid CaP, or other suitable substrate, using a coating process thatsimulates a spray granulation (or disk pelletizing) processing methodthat is well known in the art as taught by J. Reed, Principles ofCeramics Processing, Second Edition, John Wiley & Sons, Inc. New York,1995. Any form of the solid substrate, including a hollow CaPmicrosphere, is introduced in a contained system with a liquid orbinder-liquid (such as an aqueous-based acrylic emulsion) and sprayedonto the surface to promote adhesion of the loose powder or particulateonto the solid substrate surface and subsequently sintered. Theparticulate may be an agglomerate of CaP powder that contains openporosity in order to increase surface area of the CaP material forgreater activity in cell culturing applications.

[0078] A CaP coating, typical of the type described above, is notlimited in application to the surface of hollow microspheres. Calciumphosphate coatings, as formulated and tailored to specific propertyneeds, can also be applied to a variety of other substrate materialsincluding, but not limited to, alumina, mullite, cordierite, or otherceramic materials all of which are examples of oxide materials. Theporous coating can be applied to either a hollow or a solid microbead.The microbead is a substrate for bonding the porous calcium phosphatecoating in an appropriate sintering cycle. In addition to the variety ofoxide ceramic substrate materials that are available in either a hollowor solid microbead or both, other materials may be used in providing asubstrate surface for bonding the porous calcium phosphate coating.Glass beads, with or without BioGlass (L. L. Hench, Univ. of Florida),as an example, offer the advantage of softening over a broad temperaturerange. The softening characteristic of glass is useful for bonding thecalcium phosphate coating. Similarly, a polymeric substrate material,particularly one that has an amorphous phase in its structure, also mayexhibit a softening effect with temperature or solvent treatment of thesurface. This softening makes it easier to bond a calcium phosphatecoating because of the softening and wetting of the particulate calciumphosphate coating material. Examples of polymeric/organic materials thathave utility as a substrate in either a hollow or solid microsphere forminclude, but are not limited to, dextran, polyethylene, polypropylene,polystyrene, polyurethane, and collagen.

[0079] The porous structure of the calcium phosphate coating provides alarge surface area interface that is well suited for culturinganchorage-dependent cells. In certain applications, growth enhancingmaterials, such as biological coatings, including growth factors whichmay be added separately in the porous structure or coated onto thesurface of the coating, are useful in promoting cell and tissue growthwith the calcium phosphate substrate providing additional mechanical andanchorage-dependent functionality. Collagen and other bio-adhesives(coated or added separately) provide special utility in promoting celladhesion and are well suited for adapting to the coarse porosity in theCaP coating. Collagen also has the potential for forming “bridges”between individual CaP coated hollow or solid microbeads.

EXAMPLE 4 Porous Calcium Phosphate Coating for Application on CalciumPhosphate, Other Ceramic, Glass (BioGlass®), and Polymer HollowMicrospheres and Spheres

[0080] A typical example of a porous calcium phosphate coating, having afine pore size distribution, typically less than about 30 micrometers,is comprised of the following components: Volume Percent As-received orCalcined Tribasic Calcium Phosphate Powder 50-80 Corn Starch Pore Formeror Expancel 461-DE 20 20-50 Total Calcium Phosphate and Fine Pore FormerComponents 100 Aqueous Formulation Total Calcium Phosphate/Fine PoreFormer Components 25-40 Distilled Water 60-75 Ammonium PolyacrylateDispersant 0.5-1.0

[0081] A typical example of a porous calcium phosphate coating having acoarse pore size distribution with the majority of pore sizes in therange of 30-60 micrometers is comprised of the following components:Volume Percent As-received or Calcined Tribasic Calcium Phosphate Powder50-80 Potato Starch Pore Former or Expancel 091-DE 80 20-50 TotalCalcium Phosphate and Coarse Pore Former Components 100 AqueousFormulation Total Calcium Phosphate/Coarse Pore Former Components 25-40Distilled Water 60-75 Ammonium Polyacrylate Dispersant 0.5-1.0

[0082] Material Sources:

[0083] Tribasic Calcium Phosphate: FMC, Lawrence, Kans.

[0084] Corn Starch: Argo Brand, CPC Int., Inc., Englewood Cliffs, N.J.

[0085] Expancel Polymers: Expancel, Inc., Duluth, Ga.

[0086] Potato Starch: Western Polymer Corp., Moses Lake, Wash.

[0087] Polyacrylate Dispersant (Duramax D-3021): Rohm & Haas,Montgomeryville, Pa.

[0088] To reduce the surface area of the as-received tribasic calciumphosphate powder for ease of slurry processing at higher solids content,the material is calcined in the 1100-1250° C. temperature range prior tomixing with the pore former, water and dispersant components. Thepreceding porous coating formulations are sintered in the temperaturerange of about 1000° C. to 1300° C. for bonding to the surface of CaP orother ceramic spheres. For glass or polymer spheres, the coatings arebonded to the surface at a temperature near the softening point of theglass or polymer composition.

EXAMPLE 5 Aggregate Suspendable Microcarriers with Open Porosity

[0089] Microcarrier spheres are prepared as set forth above by themodified sol gel, the Torobin spray nozzle method or other methodsadapted from procedures as set forth in Hollow and Solid Microspheres(D. Wilcox, supra.) The density of the hollow microspheres is adjustedto less than 1.0 gms/cc to offset the additional fired weight of thebonding CaP medium to be added. A slurry of CaP, made either fromcommercially available powders such as FMC (Lawrence, Kans.) orsol-prepared reacted sources (see Hayek and Newesley in Jacob Kleinberg,supra.) is prepared in H₂O with a solids content of 15-35% by weight.Approximately 1% of methylcellulose (Dow Chemical, Midland, Mich.) and 6drops of Pluronic (BASF, Parsippany, N.J.) per 20 gms of CaP solids isadded to improve processing and green strength of formed aggregates. Theslurry and microspheres are mixed together in a container until theslurry covers the microspheres with a thin coating. The coated spheresare subsequently placed in a form for containment during sintering. Theform used for containment can either be thermally decomposed (papercontainer) during sintering or separated from the aggregate aftersintering in a ceramic crucible. The green aggregate is dried at 100° C.for one hour and subsequently fired at a rate of about 100° C./hr to1100-1300° C. for 1 hour to obtain composites of spheres withinterconnecting open porosity. The fired aggregate can subsequently bepulverized, sieved and classified by liquid gravity separation into thedesired size/density of aggregate which would allow for the growth ofcells, during cell culture, within protected open pores, in addition tothe outer surface of the aggregates.

EXAMPLE 6 Aggregate Suspendable Microcarriers with Closed Porosity

[0090] For a CaP hollow microsphere, the amount of enclosed porosityrequired to achieve suspendability is the range of about 60% to 70%. Forother than a hollow microsphere, this amount of porosity isredistributed in an aggregate form having multiple enclosed pores.Microcarriers are fabricated by addition of foaming agents to CaPslurries, and subsequently drying these slurries, such that the enclosedbubbles formed during foaming remain in place to provide a closedmulti-pore structure. A typical foaming agent is an organic wettingagent which in a slurry, exhibits low surface tension and is capable ofcreating a foamed structure with the mechanical shear of the slurry.Such foaming agents include, for example, a Triton X-100 surfactant(Spectrum Chemical Mfg. Corp., Gardena, Calif.). Another example of afoamed microcarrier is one created by a chemical reaction which producesgas that coalesces as bubbles within the CaP slurry (e.g., oxidation ofhydrogen peroxide) and is subsequently dried and thermally processed.

[0091] In both cases, the foamed aggregates are sintered toapproximately 1200 to 1350° C. such that interstitial CaP materialbetween the voids is impermeable to liquid media penetration. Theseaggregates are made to have neutral buoyancy in liquid media byadjusting the CaP solids to void content of the monolith. Thesematerials are subsequently ground by standard granulation techniques andclassified by screening or air classification to the desired size neededin cell culture.

EXAMPLE 7 Composite Suspendable or Non-Suspendable Microcarriers

[0092] In forming suspendable composites, the amount of CaP filleradditive that can be used relative to a polymer or glass material isdirectly related to the density of the bulk polymer or glass material.However, the solid form polymer or glass can be made to contain asignificant amount of closed porosity phase to reduce its bulk density.For polystyrene or dextran as examples of polymers exhibiting utility asa cell culture material, the presence of closed porosity created by afoaming agent which lowers the bulk density, will allow for the additionof more CaP filler in meeting the suspendability requirement due to itshigher density.

[0093] A glass or polymer hollow microsphere is the preferred compositepre-cursor. A composite structure is formed using CaP powder orparticulate as filler material. The final desired density, for achievingbuoyancy as a suspendable microcarrier, will take into account thepolymer or glass phase, CaP filler phase and a substantial pore phasethat results from the hollow microsphere-forming process. Processmethods for forming hollow microsphere diameters in the size range ofabout 100 micrometers to about 6 millimeters are useful in thisinvention. A method that allows for a substantial void (e.g., about 30%to about 60% of the overall bulk volume) in the microcarrier providesthe means for a high loading of CaP filler in forming the compositestructure. Polymeric materials that can be used include polystyrene,dextran, polyethylene, and others available in hollow microsphere form.The example below illustrates the degree of CaP loading for such apolymeric hollow microsphere. Although higher in density, a hollow glassmicrosphere is also capable of accepting CaP filler loading forachieving suspendability.

EXAMPLE 8 CaP Filler Loading in Hollow Polymeric Microsphere

[0094] For a polymer with a 1.1 gms/cc density in a hollow microsphereform with 60% void volume, the bulk density is 0.44 gms/cc. Therefore,the following equation can be written:

x(0.44 gms/cc)+(1−x)(3.0 gms/cc)=1.05 gms/cc as example of density forbuoyancy for the composite

[0095] where

[0096] x=volume fraction of polymer in hollow microsphere form

[0097] 1−x=volume fraction of CaP filler in the composite

[0098] with x=0.76 as the volume fraction of hollow polymer microsphere,the corresponding volume fraction of CaP filler is 0.24 or 24%.

[0099] Non-suspendable composites of CaP can be prepared by the additionof CaP powders and/or particulates to conventional materials used incell culture, such as polystyrene, collagen, dextran, gelatin, or glassas examples. Similar materials such as polyethylene, polyurethane, andsilicones or the like could also be used. In the case of thermoplasticpolymers, sufficient CaP could be added to the melted polymer to bondthe powders or particulates into a coherent mass of sufficient strengthto resist crumbling during cell culture. CaP particle loading ranges canbe from about 10 to 80 volume percent and are adjustable to therequirements of the culture of specific cells. Likewise, the CaPpowders/particulates can be added to liquid/collagen or gelatin slurriesin equivalent previously cited ranges of CaP volume percent sufficientto maintain mechanical integrity in cell culture environments. In theaforementioned cases, the materials can be either dried under heat orfrozen and subsequently broken up by standard means of granulation tothe desired particle sizes, or atomized by spray drying or otherconventional polymer processing techniques, as previously described, andfurther classified by standard sieving methods. A similar process couldalso be used in the case of CaP additions to glass, although the CaPmaterial would have to be added to the melted glass preferably below1300° C. The melted glass is subsequently cooled to room temperature andground/classified to size or atomized from the melt to produceparticulates, which are classified to the desired sizes. All of thesecomposites could be used as cell culture substrates or in discreteparticulate forms in cell culture-packed beds or fluidized beds thatrequire higher density as previously described. Likewise, the activityof the CaP filler can be further enhanced by abrading the surface of thecomposite structures.

EXAMPLE 9 CaP Non-Suspendable Microcarrier with Open Porosity

[0100] CaP ceramic microspheres in the range of about 0.5 mm to about 6mm in the unsintered state are produced from ceramic slurries usingeither a commercial source of starting CaP raw material (calciumphosphate tribasic) or a precipitated form of CaP based on the nitratesolution process as taught by Jacob Kleinberg, editor, InorganicSynthesis, McGraw-Hill, New York, Vol. 7, pp. 63-65 (Hayek andNewesley), 1963. Using the commercial source of CaP raw material (FMC,Lawrence, Kans.), slurry processing properties are controlled byoptimizing several variables, including slurry density-related to solidscontent, viscosity, particle size distribution, organic binders,including pore formers, and dispersing agents. Slurry processing isfollowed by spray drying which is performed using conventional methodswell known in the art as taught by K. Masters, Spray Drying, LeonardHill Books, London, England, 1972. The preferred method for producingmicrospheres with diameters greater than one (1) millimeter is by diskpelletizing which is well known in the art of ceramic processing.Methods for producing open porosity include the previously cited organicpore formers, and/or by sintering the microspheres to a temperature lessthan that required to completely density the material, e.g., about 1100°C. for the HA form of CaP. The coaxial nozzle process as set forth inExample 1 above can also be used to produced hollow microspheres withopen porosity. Open porosity is obtained by lower temperature sinteringas described above. An alternative method for fabricating ceramic hollowspheres is set forth in U.S. Pat. No. 3,875,273 by Martin and can beused to manufacture the CaP microspheres of the present invention.

EXAMPLE 10 Aggregate CaP Non-Suspendable Microcarrier with Open Porosity

[0101] Microcarrier spheres are prepared as set forth above in Example9. A slurry of CaP, made either from commercially available powders suchas from FMC (Lawrence, Kans.) or sol-prepared reacted sources (see Hayekand Newesley in Jacob Kleinberg, supra.), is prepared in H₂O with asolids content of about 10-20% by weight. Approximately 1% ofmethylcellulose (Dow Chemical, Midland, Mich.) and 6 drops of Pluronic(BASF, Parsippany, N.J.) per 20 gms of CaP solids is added to improveprocessing and green strength of formed aggregates. The slurry andmicrospheres are mixed together in a container until the slurry coversthe microspheres with a thin coating. The coated spheres aresubsequently placed in a form for containment during sintering. The formused for containment can either be thermally decomposed during sinteringor separated from the aggregate after sintering. The green aggregate isdried at 100° C. for one hour and subsequently fired at a rate of about100° C./hr to about 1100° C./hr to obtain bonded microspheres withinterconnecting open porosity. An alternative method for bondingmicrospheres to make aggregates uses a CaP cement as taught by L. Chowand S. Takagi in U.S. Pat. No. 5,525,148. The fired or cement-bondedaggregate can subsequently be pulverized, sieved and classified byliquid gravity separation into the desired size/density of aggregatewhich would allow for the growth of cells, during cell culture, withinprotected open pores, in addition to the outer surface of theaggregates.

[0102] For Examples 1-10, the CaP microcarriers can be used inconventional cell culturing systems. For example, in cell culturescomprising the BHK 21 (baby hamster kidney) cell line, BHK 21 cells areanchored to the surface of the CaP microcarrier. This cell line may beused to produce IL2 (Interleukin 2 Factor) by secretion (Cartwright,supra).

EXAMPLE 11 CaP Microspheres for Chromatographic Applications

[0103] Microspheres for this application are produced by the processingmethods set forth in Examples 1, 1A, and 2 with the exception that themicrospheres are sintered to less than full density to make openinterconnected porosity in the range of about 20% to 50% and a pore sizerange from about 0.01 to 0.5 micrometers. This open porosity is producedby sintering the microspheres at a temperature in the range of about1100° C. to 1200° C. An example of an application is the separation ofsingle- and double-stranded DNA in a HA column as taught by K. Sundaramand L. Loane, “Liquid Chromatographic Assay For The Separation ofSingle- and Double-Stranded DNA By Using UV and UV Diode-Array Detectorsand Hydroxylapatite Column”, Journal of Liquid Chromatography, 18(5),925-939 (1995).

EXAMPLE 12 Implantable CaP Hollow Microspheres

[0104] Hollow microspheres with a diameter of about 500 micrometers areproduced by methods set forth in Examples 1, 1A, and 2 with theexception that the microspheres are sintered to less than full densityto make open interconnected porosity in the range of about 20% to 30%and a pore size range from about 0.1 to 1.0 micrometers. Thesemicrospheres are placed in a solution containing transforming growthfactor—beta to infiltrate and coat the microspheres with the growthfactor for the repair of bone defects as taught by A. Ammann et al. inU.S. Pat. No. 5,422,340. The amount of growth factor in the microspherecan be increased by infiltrating under vacuum. The microspheres aresuspended in saline in a syringe and subsequently delivered to the bonedefect site. An alternative method for delivering the microspheres forimplantation is to mix the microspheres with CaP cement at the time ofimplantation. The cement as taught by L. Chow and S. Takagi in U.S. Pat.No. 5,525,148 can be used for this method.

EXAMPLE 13 Implantable CaP Aggregate with Bonded Hollow Microspheres

[0105] An aggregate for this application is produced by methods setforth in Example 10 with the exception that the aggregate is left in amonolithic form and is not ground or pulverized. The aggregate issubsequently infiltrated with the transforming growth factor—beta asdescribed in Example 12. During implantation, the monolith is carved tothe desired implant shape and inserted in the defect site.

[0106] The foregoing description of the invention is only exemplary forpurposes of illustration. Without departing from the spirit and scope ofthe invention, one skilled in the art can make changes and modificationsto the invention to adapt it to various uses and conditions. Suchchanges and modifications are within the scope of the disclosedinvention.

[0107] All documents referred to herein are incorporated by reference.

DOCUMENTS

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We claim:
 1. A hollow CaP microbead having a density about 1.01 grams/ccto about 1.12 grams/cc.
 2. A hollow microbead according to claim 1wherein the CaP comprises 0 to 100% hydroxylapatite (HA), 0 to 100%tricalcium phosphate (TCP) and/or 0 to 100% other calcium phosphatematerials.
 3. A hollow microbead according to claim 2 comprising 0 to100% hydroxylapatite (HA) and 0 to 100% tricalcium phosphate (TCP).
 4. Ahollow microbead according to claim 1 comprising a wall, wherein thewall is essentially impermeable to aqueous media.
 5. A hollow microbeadaccording to claim 1 having an average diameter in an essentiallyspherical shape of from about 100 micrometers to about 6 millimeters. 6.A hollow microbead according to claim 1 further comprising a porouscoating.
 7. A hollow microbead according to claim 1 further comprising abiological coating.
 8. A hollow microbead according to claim 7 whereinthe biological coating is a growth factor.
 9. A hollow CaP microbeadhaving a density from about 1.2 grams/cc to about 2.0 grams/cc.
 10. Ahollow microbead according to claim 9 comprising a porous coating.
 11. Ahollow microbead according to claim 9 comprising a biological coating.12. A hollow microbead according to claim 11 wherein the biologicalcoating is a growth factor.
 13. A hollow microbead according to claim 9comprising a porous coating and a biological coating.
 14. A hollowmicrobead according to claim 13 wherein the biological coating is agrowth factor.
 15. A biomedical implant comprising a microbead accordingto claims 1-14.
 16. A biomedical implant according to claim 15 furthercomprising a biological material or pharmaceutical agent.
 17. Abiomedical implant according to claim 15 having a density from about 25%to about 75% of the material's theoretical density.
 18. A biomedicalimplant according to claim 15 wherein the microbead comprises a wallwhich is essentially impermeable to aqueous media.
 19. A biomedicalimplant according to claim 15 wherein the microbead comprises a wallwhich is porous.
 20. A biomedical implant according to claim 15 whereinthe microbead comprises holes.
 21. A chromatographic column comprising ahollow microbead according to claim 9 .
 22. An aggregate comprising thehollow microbeads from claims 1 through
 13. 23. A biomedical implantcomprising an aggregate according to claim 22 .
 24. A chromatographiccolumn comprising an aggregate according to claim 22 .
 25. Hollow andsolid glass or polymer microbeads formed with or coated with particulateHA, TCP or other CaPs.
 26. Hollow and solid microbeads comprisingcomposites of HA, TCP, other CaPs and ceramic or polymeric materials.27. A microbead according to claim 26 wherein the surface of said beadis abraded.
 28. An aggregate comprising the hollow microbeads of claim25 or 27 .
 29. A biomedical implant comprising the aggregate of claim
 28. 30. A chromatographic column comprising the aggregate according toclaim 28 .
 31. Suspendable and non-suspendable aggregates comprisingmicrobeads comprising closed and/or open pores, foamed structures ofceramic, including glass, and/or polymeric composite materials.
 32. Anaggregate according to claim 28 or 31 comprising HA and TCP coatings,porous coatings, or biological coatings including growth factors.
 33. Abiomedical implant comprising an aggregate according to 28, 31 or 32.34. A method of augmenting tissue comprising implanting the biomedicalimplant of claim 33 .
 35. A biomedical implant according to claim 33 ,further comprising a biologically active agent.
 36. A biomedical implantaccording to claim 33 having a density from about 25% to about 75% ofthe material's theoretical density.
 37. A chromatographic columncomprising and aggregate according to claim 28 or 31
 38. An aggregateaccording to claim 22 that is bonded by cementatious agents.